The main goal of this Project is to study the mechanisms of cell death in mammalian neurons. Specifically, we will address the regulation of intracellular calcium concentration ([Ca2+]i) and the role of increases in intracellular free calcium in the death process of Purkinje cells studied in in vitro cerebellar slices. We propose that the process of cell death is a significant parameter in aging, and have designed a set of experiments to test the following four-part hypothesis: (1) that an increased [Ca2=]i is a causal or concomitant event in cell death; (2) that this increase is due to calcium released from subcisternal intracellular stores; (3) that hydrolysis of PDP2 and the generation of IP3 mediates such endogenous calcium release and; (4) that all or part of this calcium release process is modified in aged neurons. In all experiments data from young adult, aged and accelerated aging syndrome rates (NIA Fischer 334) will be compared. Project 1A has three areas. First we will characterize electrophysiologically the process accompanying neuronal death by investigating the in vitro electrophysiology of calcium regulation and its relation to cell death in mammalian Purkinje cells. Findings from intrasomatic and intradendritic Purkinje cell recordings will be correlated with information obtained from experiments in which the calcium-sensing dye, Fura II, is used to image changes in [Ca++]i. Patch clamp techniques will be used to evaluate the characteristics of single calcium channels. Excitotoxic agents and neurotransmitters will be applied iontophoretically or to the bath to stimulate the cell death process, 'spontaneous' cell death as it occurs in the cerebellar slices will also be studied. Project 1B will address the role of IP3 as a mediator of calcium release from intracellular stores. We will study the calcium modulatory capacity of IP3 and the consequences of inhibiting IP3 generation with phorbol ester tumor promoters and of inhibiting IP3 action by blocking IP3 receptors with heparin. Lithium will be used to augment the phosphoinositide response in (3H)-inositol-labelled cells, and the location of the hydrolyzed lipid determined by autoradiography electron microscopy. A second set of experiments will determine the distribution of IP3 under normal conditions and following several stimulation paradigms in the three groups of animals.